A Novel Cytotoxic Mechanism for Triple-Negative Breast Cancer Cells Induced by the Type II Heat-Labile Enterotoxin LT-IIc through Ganglioside Ligation.

Triple-negative breast cancer (TNBC), which constitutes 10-20 percent of all breast cancers, is aggressive, has high metastatic potential, and carries a poor prognosis due to limited treatment options. LT-IIc, a member of the type II subfamily of ADP-ribosylating-heat-labile enterotoxins that bind to a distinctive set of cell-surface ganglioside receptors-is cytotoxic toward TNBC cell lines, but has no cytotoxic activity for non-transformed breast epithelial cells. Here, primary TNBC cells, isolated from resected human tumors, showed an enhanced cytotoxic response specifically toward LT-IIc, in contrast to other enterotoxins that were tested. MDA-MB-231 cells, a model for TNBC, were used to evaluate potential mechanisms of cytotoxicity by LT-IIc, which induced elevated intracellular cAMP and stimulated the cAMP response element-binding protein (CREB) signaling pathway. To dissect the role of ADP-ribosylation, cAMP induction, and ganglioside ligation in the cytotoxic response, MDA-MB-231 cells were exposed to wild-type LT-IIc, the recombinant B-pentamer of LT-IIc that lacks the ADP-ribosylating A polypeptide, or mutants of LT-IIc with an enzymatically inactivated A1-domain. These experiments revealed that the ADP-ribosyltransferase activity of LT-IIc was nonessential for inducing the lethality of MDA-MB-231 cells. In contrast, a mutant LT-IIc with an altered ganglioside binding activity failed to trigger a cytotoxic response in MDA-MB-231 cells. Furthermore, the pharmacological inhibition of ganglioside expression protected MDA-MB-231 cells from the cytotoxic effects of LT-IIc. These data establish that ganglioside ligation, but not the induction of cAMP production nor ADP-ribosyltransferase activity, is essential to initiating the LT-IIc-dependent cell death of MDA-MB-231 cells. These experiments unveiled previously unknown properties of LT-IIc and gangliosides in signal transduction, offering the potential for the targeted treatment of TNBC, an option that is desperately needed.

Access to health knowledge for health equality: a multi-phase review focused on disability-health.

BACKGROUND: The existing evidence base indicates increased interest in knowledge translation (KT), or, the dissemination of research to ensure uptake and impact. Given this definition, this study aimed to review existing scholarship on knowledge translation (KT) of health research to people living with disabilities (PLWD), and assess the current state of accessibility of health knowledge for people living with disabilities. METHODS: Given existing heterogeneity in literature as well as a number of varying definitions for both disability and knowledge translation, a reflexive, three-phase approach was utilized to improve methodological soundness. Phase I recognizes that existing review-style studies have been conducted on disability-KT. An existing systematic review on KT specific to the field of rehabilitation and physical medicine was analyzed to assess potential best practices towards inclusivity and accessibility for people living with disability. Phase II used the Center on Knowledge Translation for Disability and Rehabilitation Research (KTDRR) database as an information-source with high-specificity to disability-health KT. Phase III sought to rapidly assess the current landscape of systematic reviews relevant to disability-health KT, with four systematic reviews meeting the inclusion criteria across Cochrane, Psycinfo, CINAHL, PubMed, Web of Science, and EMBASE. RESULTS: The current landscape of disability-health KT is primarily targeted at health professionals who serve PLWD. PLWD are included in KT, mostly as key informants, or as study participants in KT-studies designed as health interventions. Multiple systematic reviews on disability-health KT exist, presenting vastly different foci which prevent assessment of best practices. CONCLUSIONS: KT efforts are abundant and can be seen across health research related to disabilities, generating considerable literature and systematic reviews. With regards to meeting the public health objective of equalizing and enhancing access to health knowledge, future knowledge translation efforts intending to provide PLWD with up-to-date health research can be of significant value.

Persistent neurological symptoms and elevated intracranial pressures in a previously healthy host with cryptococcal meningitis.

Cryptococcal meningoencephalitis can occur in both previously healthy and immunocompromised hosts. Here, we describe a 55 year-old HIV-negative male with no known prior medical problems, who presented with three months of worsening headaches, confusion, and memory changes without fever. Magnetic resonance imaging of the brain demonstrated bilateral enlargement/enhancement of the choroid plexi, with hydrocephalus, temporal and occipital horn entrapments, as well as marked periventricular transependymal cerebrospinal fluid (CSF) seepage. CSF analysis yielded a lymphocytic pleocytosis and cryptococcal antigen titer of 1:160 but sterile fungal cultures. Despite standard antifungal therapy and CSF drainage, the patient had worsening confusion and persistently elevated intracranial pressures. External ventricular drainage led to improved mental status but only with valve settings at negative values. Ventriculoperitoneal shunt placement could thus not be considered due to a requirement for drainage into the positive pressure venous system. Due to this persistent CSF inflammation and cerebral circulation obstruction, the patient required transfer to the National Institute of Health. He was treated for cryptococcal post-infectious inflammatory response syndrome with pulse-taper corticosteroid therapy, with resultant reductions in CSF pressures along with decreased protein and obstructive material, allowing successful shunt placement. After tapering of corticosteroids, the patient recovered without sequelae. This case highlights (1) the necessity to consider cryptococcal meningitis as a rare cause of neurological deterioration in the absence of fever even in apparently immunocompetent individuals and (2) the potential for obstructive phenomena from inflammatory sequelae and the prompt response to corticosteroid therapy.

Enhancement of humoral immunity by the type II heat-labile enterotoxin LT-IIb is dependent upon IL-6 and neutrophils.

LT-IIb, a type II heat-labile enterotoxin produced by Escherichia coli, is a potent intradermal adjuvant that enhances immune responses to coadministered antigens. Although the immune mechanisms that promote this augmented immune response have not been well defined, prior intradermal immunization experiments suggested that early cellular and immunomodulatory events at the site of immunization modulated the augmentation of antigen-specific immune responses by LT-IIb. To investigate that hypothesis, mice were intradermally immunized with a recombinant ricin vaccine, a prospective toxin subunit antigen, in the presence and absence of LT-IIb. Analysis of tissue-fluid collection, coupled with histologic sections from the site of intradermal immunization, revealed that a single dose of LT-IIb induced local production of interleukin 6 and promoted a regional infiltration of neutrophils. The adjuvant effects of LT-IIb were abrogated in interleukin 6-deficient mice and when mice were depleted of neutrophils by pretreatment with anti-Ly6G. Overall, these data firmly demonstrated that LT-IIb, when used as an intradermal adjuvant, recruits neutrophils and is a potent rapid inducer of interleukin 6.

Novel Strategy To Protect against Influenza Virus-Induced Pneumococcal Disease without Interfering with Commensal Colonization.

Streptococcus pneumoniae commonly inhabits the nasopharynx as a member of the commensal biofilm. Infection with respiratory viruses, such as influenza A virus, induces commensal S. pneumoniae to disseminate beyond the nasopharynx and to elicit severe infections of the middle ears, lungs, and blood that are associated with high rates of morbidity and mortality. Current preventive strategies, including the polysaccharide conjugate vaccines, aim to eliminate asymptomatic carriage with vaccine-type pneumococci. However, this has resulted in serotype replacement with, so far, less fit pneumococcal strains, which has changed the nasopharyngeal flora, opening the niche for entry of other virulent pathogens (e.g., Streptococcus pyogenes, Staphylococcus aureus, and potentially Haemophilus influenzae). The long-term effects of these changes are unknown. Here, we present an attractive, alternative preventive approach where we subvert virus-induced pneumococcal disease without interfering with commensal colonization, thus specifically targeting disease-causing organisms. In that regard, pneumococcal surface protein A (PspA), a major surface protein of pneumococci, is a promising vaccine target. Intradermal (i.d.) immunization of mice with recombinant PspA in combination with LT-IIb(T13I), a novel i.d. adjuvant of the type II heat-labile enterotoxin family, elicited strong systemic PspA-specific IgG responses without inducing mucosal anti-PspA IgA responses. This response protected mice from otitis media, pneumonia, and septicemia and averted the cytokine storm associated with septic infection but had no effect on asymptomatic colonization. Our results firmly demonstrated that this immunization strategy against virally induced pneumococcal disease can be conferred without disturbing the desirable preexisting commensal colonization of the nasopharynx.

The Divergent CD8+ T Cell Adjuvant Properties of LT-IIb and LT-IIc, Two Type II Heat-Labile Enterotoxins, Are Conferred by Their Ganglioside-Binding B Subunits.

Poor immune responses elicited by vaccine antigens can be enhanced by the use of appropriate adjuvants. Type II heat-labile enterotoxins (HLT) produced by Escherichia coli are extremely potent adjuvants that augment both humoral and cellular immunity to co-administered antigens. Recent findings demonstrate that LT-IIb and LT-IIc, two type II HLT adjuvants, exhibit potent, yet distinguishable CD8(+) T cell adjuvant properties. While LT-IIc elicits a robust and rapid response at one week after administration, LT-IIb engenders a more gradual and slower expansion of antigen-specific CD8(+) T cells that correlates with improved immunity. The variations in immune effects elicited by the HLT adjuvants have been generally attributed to their highly divergent B subunits that mediate binding to various gangliosides on cell surfaces. Yet, HLT adjuvants with point mutations in the B subunit that significantly alter ganglioside binding retain similar adjuvant functions. Therefore, the contribution of the B subunits to adjuvanticity remains unclear. To investigate the influence of the B subunits on the enhancement of immune responses by LT-IIb and LT-IIc, chimeric HLT were engineered in which the B subunits of the two adjuvants were exchanged. Comparing the immune potentiating characteristics of both native and chimeric HLT adjuvants, it was found that not all the adjuvant characteristics of the HLT adjuvants were modulated by the respective B subunits. Specifically, the differences in the CD8(+) T cell kinetics and protective responses elicited by LT-IIb and LT-IIc did indeed followed their respective B subunits. However, induction of IL-1 from macrophages and the capacity to intoxicate cells in a mouse Y1 adrenal cell bioassay did not correlate with the B subunits. Therefore, it is likely that additional factors other than the B subunits contribute to the effects elicited by the HLT adjuvants.

Intradermal administration of the Type II heat-labile enterotoxins LT-IIb and LT-IIc of enterotoxigenic Escherichia coli enhances humoral and CD8+ T cell immunity to a co-administered antigen.

Vaccinations are extremely effective at combating infectious diseases. Many conserved antigen (Ag) targets, however, are poorly immunogenic. Protein subunit vaccines frequently elicit only humoral immune responses and fail to confer protection against serious intracellular pathogens. These barriers to vaccine development are often overcome by the use of appropriate adjuvants. Heat-labile enterotoxins (HLT) produced by enterotoxigenic strains of Escherichia coli are potent adjuvants when administered by mucosal or systemic routes. The efficacy of the type II HLT, however, has not been well-defined when administered by the intradermal (ID) route. Using a murine ID immunization model, the adjuvant properties of LT-IIb and LT-IIc, two type II HLTs, were compared with those of LT-I, a prototypical type I HLT. While all three HLT adjuvants enhanced Ag-specific humoral responses to similar levels, LT-IIb and LT-IIc, in contrast to LT-I, induced a more vigorous Ag-specific CD8+ T cell response and proffered faster clearance of Listeria monocytogenes in a challenge model. Additionally, LT-IIb and LT-IIc induced distinct differences in the profiles of the Ag-specific CD8+ T cell responses. While LT-IIc stimulated a robust and rapid primary CD8+ T cell response, LT-IIb exhibited slower CD8+ T cell expansion and contraction kinetics with the formation of higher percentages of effector memory cells. In comparison to LT-I and LT-IIc, LT-IIb evoked better long-term protection after immunization. Furthermore, LT-IIb and LT-IIc enhanced the total number of dendritic cells (DC) in the draining lymph node (DLN) and expression of costimulatory molecules CD80, CD86, and CD40 on DCs. In contrast to LT-I, LT-IIb and LT-IIc induced less edema, cellular infiltrates, and general inflammation at the site of ID injection. Thus, LT-IIb and LT-IIc are attractive comprehensive ID adjuvants with unique characteristic that enhance humoral and cellular immunity to a co-administered protein Ag.

LT-IIb(T13I), a non-toxic type II heat-labile enterotoxin, augments the capacity of a ricin toxin subunit vaccine to evoke neutralizing antibodies and protective immunity.

Currently, there is a shortage of adjuvants that can be employed with protein subunit vaccines to enhance protection against biological threats. LT-IIb(T13I) is an engineered nontoxic derivative of LT-IIb, a member of the type II subfamily of heat labile enterotoxins expressed by Escherichia coli, that possesses potent mucosal adjuvant properties. In this study we evaluated the capacity of LT-IIb(T13I) to augment the potency of RiVax, a recombinant ricin toxin A subunit vaccine, when co-administered to mice via the intradermal (i.d.) and intranasal (i.n.) routes. We report that co-administration of RiVax with LT-IIb(T13I) by the i.d. route enhanced the levels of RiVax-specific serum IgG antibodies (Ab) and elevated the ratio of ricin-neutralizing to non-neutralizing Ab, as compared to RiVax alone. Protection against a lethal ricin challenge was also augmented by LT-IIb(T13I). While local inflammatory responses elicited by LT-IIb(T13I) were comparable to those elicited by aluminum salts (Imject®), LT-IIb(T13I) was more effective than aluminum salts at augmenting production of RiVax-specific serum IgG. Finally, i.n. administration of RiVax with LT-IIb(T13I) also increased levels of RiVax-specific serum and mucosal Ab and enhanced protection against ricin challenge. Collectively, these data highlight the potential of LT-IIb(T13I) as an effective next-generation i.d., or possibly i.n. adjuvant for enhancing the immunogenicity of subunit vaccines for biodefense.

LT-IIc, a new member of the type II heat-labile enterotoxin family encoded by an Escherichia coli strain obtained from a nonmammalian host.

Two families of bacterial heat-labile enterotoxins (HLTs) have been described: the type I HLTs are comprised of cholera toxin (CT) of Vibrio cholerae, LT-I of Escherichia coli, and several related HLTs; the type II HLTs are comprised of LT-IIa and LT-IIb. Herein, we report LT-IIc, a new type II HLT encoded from an enterotoxigenic E. coli (ETEC) strain isolated from an avian host. Using a mouse Y1 adrenal cell bioassay, LT-IIc was shown to be less cytotoxic than CT, LT-IIa, or LT-IIb. Cytotoxicity of LT-IIc was partially neutralized by antisera recognizing LT-IIa or LT-IIb but not by anti-CT antiserum. Genes encoding putative A polypeptide and B polypeptides of LT-IIc were arranged in an operon which was flanked by potential prophage sequences. Analysis of the nucleotide and predicted amino acid sequences demonstrated that the A polypeptide of LT-IIc has moderate homology to the A polypeptides of CT and LT-I and high homology to the A polypeptides of LT-IIa and LT-IIb. The B polypeptide of LT-IIc exhibited no significant homology to the B polypeptides of CT and LT-I and only moderate homology to the B polypeptides of LT-IIa and LT-IIb. The binding pattern of LT-IIc for gangliosides was distinctive from that of either LT-IIa or LT-IIb. The data suggest that other types of the type II HLT subfamily are circulating in the environment and that host specificity of type II HLT is likely governed by changes in the B polypeptide which mediate binding to receptors.

Protein arginine methylation in health and disease.

Protein arginine methylation is a rapidly growing field of biomedical research that holds great promise for extending our understanding of developmental and pathological processes. Less than ten years ago, fewer than two dozen proteins were verified to contain methylarginine. Currently, however, hundreds of methylarginine proteins have been detected and many have been confirmed by mass spectrometry and other proteomic and molecular techniques. Several of these proteins are products of disease genes or are implicated in disease processes by recent experimental or clinical observations. The purpose of this chapter is twofold; (1) to re-examine the role of protein arginine methylation placed within the context of cell growth and differentiation, as well as within the rich variety of cellular metabolic methylation pathways and (2) to review the implications of recent advances in protein methylarginine detection and the analysis of protein methylarginine function for our understanding of human disease.